title: β-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.
abstract: The gene encoding a thermostable β-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-β-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.
authors.affiliation: Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jember, Jalan Kalimantan 37, Jember, 68121, Indonesia.
authors.forename: Anak Agung Istri
authors.initials: AA
authors.lastname: Ratnadewi
authors.forename: Muchzainal
authors.initials: M
authors.lastname: Fanani
authors.forename: Sari Dewi
authors.initials: SD
authors.lastname: Kurniasih
authors.forename: Makiko
authors.initials: M
authors.lastname: Sakka
authors.forename: Eddy Bagus
authors.initials: EB
authors.lastname: Wasito
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakka
authors.forename: Zeily
authors.initials: Z
authors.lastname: Nurachman
authors.forename: Ni Nyoman Tri
authors.initials: NN
authors.lastname: Puspaningsih
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D056506:Geobacillus / Q000145:classification* / Q000201:enzymology*; D008969:Molecular Sequence Data; D013045:Species Specificity; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase